A composition of yeast extract and yeast for obtaining the same, and a process for production of the composition of yeast extract.
This invention relates to a composition of yeast extract having taste, of kombu (Laminariales, kelps) and mushrooms and flavor thereof, new yeast strain for obtaining the same, and a process for production of the composition of yeast extract.
Conventionally used extracts of mushrooms and kombu (Laminariales, kelps, hereinafter designated as kombu) have good flavors, but addition of these natural extracts to the foods can only show simple taste. In order to increase richness in flavor, heaviness of the taste and better taste, various seasonings have simultaneously to be added. Consequently, the said extracts are not satisfactory for the purpose of commonly used or all-purpose seasonings.
Recently, the use of natural seasonings is being recognized once again according to the natural food-oriented minds of the general consumers. For expanding applicable fields of natural seasonings, yeast extracts have widely been used, and demands for higher level of quality of the yeast extracts are increasing.
Generally, raw materials of yeast extracts include genus Saccharomyces such as baker""s yeast and brewery yeast. The yeast extracts from Saccharomyces yeast have a specific yeast flavor. The yeast flavor is preferable for increasing meat taste, but is disadvantageous for taste of Japanese style cuisine.
The yeast extracts from genus Candida (hereinafter designated as Candida yeast extracts) have almost no yeast flavor which is specific to yeast extracts originated from yeast belonging to genus Saccharomyces. Furthermore, Candida extract can be used in all-purpose seasonings preferable to the taste of Japanese cuisine having savory (umami) by using nucleic acids which are rich in Candida yeast. (Japanese Patent Unexamined Publication No. 62-201595), Japanese Patent Examined Publications No. 7-93871 and No. 56-46824).
These prior technologies have objective to increase the taste of nucleic acids. For this purpose, the yeast is treated in order to increase the content of nucleic acids by enzymatic decomposition. Consequently, the content of free amino acids is relatively low and this gives simple taste and flavor.
In order to improve this disadvantage, the invention of Japanese Pat. Unexam. Publ. No. 9-294581 discloses an increased intracellular glutamic acid content by obtaining yeast belonging to genus Saccharomyces which is resistant to glutamic acid antagonist. The yeast extract of the invention is, however, not able to improve flavor due to removing Saccharomyces yeast flavor.
For masking undesirable yeast flavor of the yeast extracts and adding specific good flavor to foods and cooking, a method is conventionally practiced which adds flavors such as dried bonito flavor and smoke flavor, other than yeast extract whose origin is not yeast extracts. This method is, however, complicated due to processing and causes higher production cost. Consequently, it is preferable for the yeast itself to have a specific good flavor in yeast. No trials have been made to add or to generate specific good flavor and taste in the composition of Candida yeast extract per se.
Furthermore, a method for preparing sea tangle (kombu)-like taste using sugar alcohol, potassium and glutamic acid is known (Japanese Pat. Exam. Publ. No. 6-75479 and ibid. 7-114646). This method is only a combination of chemical compounds for preparing a taste composition, and the resulting seasonings are different from the natural flavor. Therefore, the method can not provide a seasoning with a natural complex flavor. Consequently, in order to prepare sea tangle (kombu)-like natural flavor, sea tangle (kombu) extract and/or residue of sea tangle (kombu) extract should be added.
As a result of extensive studies in order to solve these problems, we have found that Candida yeast having both aspartate hydroxamate resistance and low temperature sensibility, could provide yeast extracts which contain over a certain content of mannitol, glutamic acid, alanine, 5xe2x80x2-IMP and 5xe2x80x2-GMP. We have also found that the said yeast extract showed strong taste of sea tangle (kombu) and mushrooms-like taste, and have completed the present invention.
An object of the present invention is to provide a composition of yeast extract from genus Candida comprising contents in a dried yeast extract being mannitol above 0.5%, glutamic acid above 2.0%, alanine above 0.5%, 5xe2x80x2-IMP above 1.5% and 5xe2x80x2-GMP above 1.5%.
Another object of the present invention is to provide a yeast from genus Candida resistant to aspartate hydroxamate and taking within 48 hours to grow colonies at 30xc2x0 C. on a nutrient agar medium, but taking over 96 hours to grow colonies at 15xc2x0 C.
Further object of the present invention is to provide Candida utilis AHR21 (FERM BP-6099).
A still further object of the present invention is to provide a process for production of a composition of yeast extract comprising culturing said yeast in a medium containing molasses as a main carbon source, isolating the cultured yeast, disrupting the yeast cells and treating the thus obtained destructed cell suspension with enzyme.
The present invention will be explained in detail herein below.
In the yeast extract of the present invention, contents of mannitol, glutamic acid, alanine, 5xe2x80x2-IMP and 5xe2x80x2-GMP are essentially above 0.5%, above 2.0%, above 0.5%, above 1.5% and above 1.5%, respectively. These are preferably above 1.0%, above 3.0%, above 1.0%, above 2.0% and above 2.0%, respectively.
Candida yeast extracts prepared by a conventional enzymatic digestion method have been known to contain glutamic acid, alanine and 5xe2x80x2-1 G. However, the fact that coexistence of over a certain level of glutamic acid, alanine and 5xe2x80x2-1 G with mannitol in Candida yeast can produce sea tangle (kombu)-like or mushroom-like taste, has not been known.
We have found that when the above concentration of each component is simultaneously satisfied, sea tangle (kombu)-like and mushroom-like taste and flavor are felt strongly and with good balance. Namely, addition of nuclease to the yeast which contains mannitol, glutamic acid and alanine may result to enhance taste of glutamic acid with coexisting alanine, 5xe2x80x2-IMP and 5xe2x80x2-GMP, thus to produce sea tangle (kombu)-like taste.
In this invention, a taste means, when the extract is held in the mouth, feelings of full body of the taste or spread of the taste, for example, richness or thickness in taste. A flavor means, when the extract is held in the mouth, feelings of first taste and flavor in the tongue and the nose.
A process for production of a yeast extract of the present invention is explained hereinbelow.
A yeast derived from genus Candida which is resistant to aspartate hydroxamate and takes within 48 hours to grow colonies at 30xc2x0 C. on a nutrient agar medium, but takes over 96 hours to grow colonies at 15xc2x0 C., is obtained. A mother strain of the yeast from genus Candida is preferably Candida tropicalis, Candida lypolitica and Candida utilis. Most preferable strain is Candida utilis IFO 0626. These yeasts can be obtained from Institute for Fermentation Osaka, Japan.
(1) [Production of a Strain Resistant to Aspartate Hydroxamate]
Cultured broth of genus Candida is treated by mutagen, such as nitrosoguanidine, UV or X-ray irradiation to produce survival rate of yeast about 0.01-1%. Thus obtained mutant of yeast resistant to aspartate hydroxamate is selected. The yeast strain resistant to aspartate hydroxamate can be selected by the following procedure. The mutant yeast hereinabove is cultured in complete synthetic medium (glucose 1%, ammonium sulfate 0.35%, potassium monophosphate 0.1%, magnesium sulfate 0.05%, sodium chloride 0.05%, calcium chloride 0.05%, and trace metal ions and vitamins), which contains aspartate hydroxamate about 500-1000 xcexcg/ml, then colonies having a diameter of 2-fold larger size than the diameter of the colonies of aspartate hydroxamate sensitive strains (mother strains), are selected. Subsequently, in these aspartate hydroxamate resistant strains, yeast which contains more than 2-fold amount of intercellular alanine (above 0.2% in the yeast extract) than the original strains is selected. The thus obtained alanine and proline accumulated strain is called as AHR3C-2.
{circle around (2)} [Collection of Low Temperature Sensitive Strain]
Mutational treatment is performed as same as in the above procedure. Among the obtained mutant yeasts, yeast strains, which takes within 48 hours to grow colonies at 30xc2x0 C. on a nutrient agar medium, but takes over 96 hours to grow colonies at 15xc2x0 C., i.e. yeast having low temperature sensitivity, are selected.
Selection of low temperature sensitive yeast is performed as follows.
(1) Mutant aspartate hydroxamate resistant strains are suspended in water. The suspended strain is inoculated in YPD (Yeast Peptone Dextrose) medium and cultured at 30xc2x0 C. for 18-24 hours.
(2) Cultured yeast is harvested from the cultured medium, and yeast is washed with aseptic water, then the yeast is cultured with MM medium (Bact Yeast Nitrogen Base w/o Amino acid) for 18-24 hours.
(3) Harvested yeast cells are cultured in MM medium with ammonium sulfate at 15xc2x0 C.
(4) After 6 hours of cultivation, Nystatin is added thereto to 10 xcexcg/ml and the yeast is further cultured at 15xc2x0 C. for 2 hours.
(5) The thus obtained yeast is harvested from the medium, washed with aseptic water, and reinoculated in YPD medium.
The above procedures (1)-(5) are repeated 2-3 times. The obtained yeast is washed with aseptic water and cultured on YPD agar medium. The strain which takes within 48 hours to grow colonies at 30xc2x0 C., but takes over 96 hours to grow colonies at 15xc2x0 C. is selected.
{circle around (3)} [Selection of Strain with High Content of 5xe2x80x2-IMP and 5xe2x80x2-GMP]
The thus obtained yeast is cultured in YPD medium 50 ml at 30xc2x0 C. for 18 hours. Harvested yeast cells are disrupted by using beads homogenizer. Endoprotease is added to the disrupted cell mixture and treated at 50xc2x0 C. for 12 hours. Nuclease is further added after the endoprotease treatment and is treated at 65xc2x0 C. for 3 hours. Deaminase is subsequently added and reacted at 40xc2x0 C. for 2 hours. The residue in the obtained solution is removed by centrifugation. The supernatant solution is analyzed for sugar, alcohol, amino acids and nucleic acids. From the solution, strains having more than 1.2 fold of contents of 5xe2x80x2-IMP and 5xe2x80x2-GMP comparing to that of mother strain are selected.
Mutant which can be used as yeast material for yeast extract having simultaneously satisfying amounts of mannitol, glutamic acid, alanine, 5xe2x80x2-IMP and 5xe2x80x2-GMP being above 0.5%, 2.0%, 0.5%, 1.5% and 1.5% respectively, can be obtained by the above mentioned method. An example of yeast is Candida utilis AHR21. The strain has been deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken, Japan, on Sep. 10, 1997, as permanent deposition No. FERM BP-6099.
A process for production of a composition of yeast extract using said mutant yeast is illustrated hereinbelow.
A yeast strain having the properties of the present invention is cultured and is inactivated to prepare a suspension. Examples of carbon sources of culture medium are glucose, saccharide, acetic acid, ethanol, molasses, wood sugar, dextrose corn syrup and sulphite pulp waste liquid. Among them, molasses is preferable in the point that a flavor of substance in the molasses or substances obtained from converted substance in the molasses during fermentation are effective to induce not only sea tangle (kombu)-like taste but also generate sea tangle (kombu)-like flavor.
Examples of nitrogen sources of culture medium are urea, ammonia, ammonium sulfate, ammonium chloride and nitrate. The examples of phosphate, potassium or magnesium sources can be materials which usually are used in industrial purposes. Other components, for example inorganic salts such as zinc, copper, manganese or iron ions; vitamins; amino acids; or nitrogen containing substance such as corn steep liquor can be added. Cultivation temperature may be at 20xc2x0 C.-38xc2x0 C., most preferably at 30xc2x0 C.-36xc2x0 C., and pH may be pH 3.5-8.0, more preferably at 4.0-6.0.
Cultured yeast is harvested from the medium and washed with water. Washed wet yeast cells and water are mixed as to form ratio from 2:1 to 1:2 by mixing with water to obtain suspension. The said suspension is boiled at 90-110xc2x0 C. or 20-60 minutes, or spray-dried with inlet temperature at 120-130xc2x0 C. and outlet temperature at 80-110xc2x0 C. of the atomizer in the spray drier to prepare dried yeast cells, then the yeast is inactivated.
When the yeast suspension is boiled, inactivated yeast is disrupted by physical means such as homogenizer. When the suspension is applied for spray drying, aqueous suspension of yeast, in which solid parts is set to 10-30%, is prepared. The suspension is treated with enzyme.
Neutral endoprotease is added to the suspension at 100 mg/l, and then treated at 45-55xc2x0 C. for 6-12 hours. Subsequently, nuclease is added at 30 mg/l and treated at 60-70xc2x0 C. for 2-4 hours. Further, deaminase is added at 30 mg/l and treated at 35-45xc2x0 C. for 1-2 hours.
After enzymatic treatment, residue in the suspension is removed off by centrifugation to the obtained clear solution, If necessary sodium chloride and other materials can be added. Subsequently the mixture is concentrated to desired concentration to obtain the yeast extract. Water content of the yeast extract is preferably 25-35% in the paste product and solid material of the extract is preferably 50-60%.